Kobayashi N, Katsumata H, Katayama H, Oiwa H, Goto J, Takeuchi Y
Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan.
J Steroid Biochem Mol Biol. 2000 Apr;72(5):265-72. doi: 10.1016/s0960-0760(00)00032-7.
Sulfation of the 3-hydroxy group is assumed to be a major metabolic route of ursodeoxycholic acid (UDCA) which is used for treating various hepatobiliary diseases. We have developed a sensitive enzyme-linked immunosorbent assay (ELISA) for determining the total amount of nonamidated, glycine- and taurine-amidated ursodeoxycholic acid 3-sulfates (UDCA 3-Suls) using a newly established monoclonal antibody. In this study, 12 kinds of antibody-secreting hybridoma clones were generated by a fusion experiment between P3/NS1/1-Ag4-1 myeloma cells and the spleen cells from a BALB/c or an A/J mouse which had been immunized with a conjugate of nonamidated UDCA 3-Sul and bovine serum albumin. One of the monoclonal antibodies, Ba-10 (gamma2a, kappa), had suitable binding properties for clinical application, which was group-specific to the UDCA 3-Suls, and showed negligible cross-reactivities with various related bile acids including potentially interfering compounds, namely, the unconjugated UDCA, UDCA 7-N-acetylglucosaminide, the 3-sulfates of cholic acid, chenodeoxycholic acid and deoxycholic acid. The antibody Ba-10 allowed us to develop a sensitive competitive ELISA system whose measurable range was approximately 2-200 pg per assay. A serial dilution study indicated that the ELISA enables the direct measurement of the UDCA 3-Suls in human urine before and after the administration of exogenous UDCA. The daily urinary excretion rate of UDCA 3-Suls from healthy male volunteers (n = 5) was determined to be a mean of 131 +/- 61.2 (SD) microgram as the nonamidated UDCA 3-Sul equivalent.
3-羟基硫酸化被认为是熊去氧胆酸（UDCA）的主要代谢途径，UDCA用于治疗各种肝胆疾病。我们利用新建立的单克隆抗体开发了一种灵敏的酶联免疫吸附测定法（ELISA），用于测定非酰胺化、甘氨酸酰胺化和牛磺酸酰胺化的熊去氧胆酸3-硫酸盐（UDCA 3-Suls）的总量。在本研究中，通过P3/NS1/1-Ag4-1骨髓瘤细胞与用非酰胺化UDCA 3-Sul与牛血清白蛋白的缀合物免疫的BALB/c或A/J小鼠的脾细胞之间的融合实验，产生了12种分泌抗体的杂交瘤克隆。其中一种单克隆抗体Ba-10（γ2a，κ）具有适合临床应用的结合特性，它对UDCA 3-Suls具有组特异性，并且与各种相关胆汁酸（包括潜在干扰化合物，即未结合的UDCA、UDCA 7-N-乙酰葡糖胺、胆酸、鹅去氧胆酸和脱氧胆酸的3-硫酸盐）的交叉反应可忽略不计。抗体Ba-10使我们能够开发一种灵敏的竞争性ELISA系统，其可测量范围约为每次测定2-200 pg。系列稀释研究表明，该ELISA能够直接测量外源性UDCA给药前后人尿中的UDCA 3-Suls。健康男性志愿者（n = 5）的UDCA 3-Suls每日尿排泄率经测定，以非酰胺化UDCA 3-Sul当量计平均为131 +/- 61.2（SD）微克。
